金黄色葡萄球菌荧光定量PCR检测方法的建立及其在大鼠、小鼠粪便检测中的应用

doi:10.12300/j.issn.1674-5817.2023.022

摘要/Abstract

摘要：

目的建立一种快速灵敏的金黄色葡萄球菌（Staphylococcus aureus）检测方法。方法选择金黄色葡萄球菌特异性基因nuc作为靶基因，设计并合成一对特异性引物和一条TaqMan探针，利用荧光定量PCR技术建立nuc基因的核酸检测方法，并在大鼠、小鼠粪便样本检测中进行应用。结果对金黄色葡萄球菌和其他非金黄色葡萄球菌菌株中提取的DNA进行荧光定量PCR检测，结果显示金黄色葡萄球菌出现特异性扩增曲线，而其他非金黄色葡萄球菌未出现，表明设计的引物和探针对金黄色葡萄球菌检测具有特异性。将提取的金黄色葡萄球菌DNA进行10倍梯度稀释后测定其灵敏度，结果显示最低检出限是10 fg的DNA量，比普通PCR方法高2个数量级。本研究共检测91份样品，有4份来自同一设施的大鼠样品，扩增曲线为典型的S曲线；将该PCR产物测序并进行BLAST比对，该样本的基因序列与金黄色葡萄球菌的基因序列相似度为100%，表明该样本为金黄色葡萄球菌nuc基因核酸阳性，阳性率为4.40%，与细菌培养法的检测结果一致。核酸提取采用全自动核酸纯化仪，从核酸提取到检测结果判定快速，所需时间小于1.5 h。结论建立的以nuc为靶基因鉴定金黄色葡萄球菌的qPCR方法，具有快速、灵敏度高和特异性强的优点，可用于实验动物大鼠、小鼠粪便中金黄色葡萄球菌的检测。

关键词: 金黄色葡萄球菌, 检测方法, 荧光定量PCR, 粪便, 大鼠, 小鼠

Abstract:

Objective To establish a method for rapid and sensitive detection of Staphylococcus aureus. Methods The specific gene nuc of Staphylococcus aureus was selected as the target gene. A pair of specific primers and a TaqMan probe were designed and synthesized according to the published sequence of the nuc gene. Establish a nucleic acid detection method for nuc gene using fluorescence quantitative PCR technology, and apply it clinically in the detection of fecal samples from rats and mice. Results The DNA extracted from Staphylococcus aureus and other non-Staphylococcus aureus strains was detected by qPCR. The results showed that Staphylococcus aureus had a specific amplification curve, while other non-Staphylococcus aureus did not, indicating that the designed primers and probes were specific for Staphylococcus aureus. The sensitivity of this method was determined by diluting the DNA of Staphylococcus aureus by 10 times. The results showed that the detection limit of this method was 10 fg DNA, which was 2 orders of magnitude higher than that of ordinary PCR method. A total of 91 clinical samples were detected in this study, of which 4 rat samples from the same facility had a typical S-curve. The PCR products were sequenced and BLAST compared. The gene sequence of this sample was 100% similar to that of Staphylococcus aureus, indicating that the sample was positive for the nucleic acid of Staphylococcus aureusnuc gene, with a positive rate of 4.40%. The result was consistent with that obtained by bacterial culture method. The nucleic acid extraction adopted a full-automatic nucleic acid purification instrument, and the time required from nucleic acid extraction to detection result determination was less than 1.5 h. Conclusion The qPCR method established in this study to identify Staphylococcus aureus with nuc gene as the target gene has the advantages of fast, high sensitivity and specificity, and can be used for the detection of Staphylococcus aureus in feces of rats and mice.

Key words: Staphylococcus aureus, Method of detection, Fluorescent quantitative PCR, Feces, Rats, Mice

中图分类号:

Q95-33

于灵芝,谢建芸,冯丽萍,等. 金黄色葡萄球菌荧光定量PCR检测方法的建立及其在大鼠、小鼠粪便检测中的应用[J]. 实验动物与比较医学, 2023, 43(5): 566-573. DOI: 10.12300/j.issn.1674-5817.2023.022.

Lingzhi YU,Jianyun XIE,Liping FENG,et al. Establishment of Fluorescence qPCR Method for Detection of Staphylococcus Aureus and Its Application in Feces Detection of Rats and Mice[J]. Laboratory Animal and Comparative Medicine, 2023, 43(5): 566-573. DOI: 10.12300/j.issn.1674-5817.2023.022.

图/表 6

注：红色S型曲线代表阳性对照。RFU是相对荧光单位。
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图1 金黄色葡萄球菌实时荧光定量PCR特异性检测结果
注：红色S型曲线代表阳性对照。RFU是相对荧光单位。

Figure 1 Specificity for Staphylococcus aureus detected by real-time fluorescent quantitative PCR
Note：Red S-type curve represents the positive control.RFU， Relative fluorescence unit.

注：从左向右的曲线依次代表的核酸量为1 ng、100 pg、10 pg、1 pg、100 fg、10 fg和1 fg。
">

图2 金黄色葡萄球菌进行实时荧光定量PCR的标准曲线（A）和灵敏度检测结果（B）
注：从左向右的曲线依次代表的核酸量为1 ng、100 pg、10 pg、1 pg、100 fg、10 fg和1 fg。

Figure 2 Standard curve （A） and sensitivity （B） for Staphylococcus aureus detected by real-time fluorescent quantitative PCR
Note：The amounts of nucleic acid represented by the curves from left to right is 1 ng， 100 pg， 10 pg， 1 pg， 100 fg， 10 fg and 1 fg， respectively.

表1 组内重复性实验数据（Ct值）及变异系数（CV）

Table 1 Intragroup repeatability data (Ct) and coefficient of variation (CV)

DNA量

DNA quantities

10次检测结果（Ct值）

10 test results (Ct value)

CV值/%

CV value/%

表2 组间重复性实验数据（Ct值）及变异系数（CV）

Table 2 Intergroup repeatability data (Ct) and coefficient of variation（CV）

DNA量 DNA quantities	5次检测结果（Ct值） 5 test results(Ct value)					DNA量 DNA quantities	5次检测结果（Ct值） 5 test results(Ct value)
DNA量 DNA quantities	1	2	3	4	5	DNA量 DNA quantities	1	2	3	4	5
10 pg	33.13	34.08	36.00	34.19	33.02	10 fg	26.04	26.10	26.20	26.12	26.08
	34.44	32.27	33.47	34.04	34.56		25.80	25.89	25.83	26.15	26.10
	32.72	32.60	32.35	31.90	32.56		26.17	25.39	25.86	25.76	25.91
	32.13	32.88	34.21	33.14	32.90		25.87	25.96	25.86	25.90	25.66
	33.83	35.05	32.60	33.99	33.41		25.86	25.78	26.10	26.12	25.69
平均数1 Mean 1	33.25	33.38	33.73	33.45	33.29		25.95	25.82	25.97	26.01	25.89
平均数2 Mean 2	33.42						25.93
标准差SD	0.19						0.19
CV/%	0.56						0.75

注：红色S型曲线代表扩增阳性对照，黑色S型曲线代表核酸提取阳性对照，蓝色S型曲线代表阳性样本。
">

图3 金黄色葡萄球菌实时荧光定量PCR的临床检测结果
注：红色S型曲线代表扩增阳性对照，黑色S型曲线代表核酸提取阳性对照，蓝色S型曲线代表阳性样本。

Figure 3 Clinical detection results for Staphylococcus aureus by real-time fluorescent quantitative PCR detection
Note：The red S curve represents the positive control for amplification， the black S curve represents the positive control for nucleic acid extraction， and the four blue S curves represent the positive samples.

图4 PCR产物测序序列（A）和BLAST比对结果（B）

Figure 4 Sequence of PCR product （A） and the result of BLAST （B）

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